The conformational dynamics of a biologically active, non-covalent complex containing ferro heme fragment (1-25)H and apofragments (28-38) and (3H)(56-104)(or (3H)(39-104)) of horse cytochrome c has been studied. The ferrous three-fragment complex exhibits a higher frequency of dissociation-association with fragment (28-38) and a lower frequency of overall unfolding-folding. In the presence of excess of free (28-38) and below 30 degrees C unfolding of the ferrous three-fragment complex occurs mostly without going through complex (1-25)H:(56-104) which is an intermediate for folding. Above 30 degrees C the unfolding via complex (1-25)H:(56-104) becomes detectable. The ground state of the complex appears to be maintained by a cooperative mode of interatomic interactions operative throughout the three-dimensional structure which may be disrupted upon activation. The results of chemical substitution of evolutionarily invariant Leu 32 and variant Leu 35 of (28-38) have implicated that such global cooperative interactions would be disrupted if Leu 32 is substituted by Ile, Val or Phe but maintained if Leu 35 is replaced by Ile. This would give the molecular basis for the evolutionary invariance of Leu 32.